The objective of this project is to develop recombinant HIV systems that allow the rapid quantitation and analysis of drug-resistant 'breakthrough' HIV infection of host cells. These model systems will be used in the pre-clinical assessment of antiretrovirals to identity compounds or combinations of compounds that minimize or eliminate breakthrough HIV infection. Information derived from these model systems may lead to the design of more effective therapies for HIV-1 infection. The specific aims of this project are to: 1.1 Produce high-titer recombinant HIVs encoding reporter genes that allow the rapid identification of individual virus-infected cells. 1.2 Use these recombinant HIVs to quantitate the frequency of breakthrough HIV infection of host cells in the presence of different antiretroviral compounds and combinations of compounds. 1.3. Analyze the mechanism underlying breakthrough infection, differentiating HIV-encoded drug resistance from cellular-encoded drug resistance. 1.4 Refine our existing retroviral systems to rapidly define the genetics of individual virus-encoded drug-resistant infectious events. 1.5. Quantitate the prevalence of drug-resistant viral variants within unselected virus populations (laboratory strains and clinical isolates). 1.6 Develop similar recombinant retroviral systems to study resistance to antiviral agents that inhibit later stages of the HIV replicative cycle. Preliminary studies with recombinant HIV encoding guanine phosphoribosyltransferase (HIV-gpt) have demonstrated that this approach is feasible and that rare (less than 10[-4]) drug-resistant infection of individual host cells can be detected and quantitated. Construction of recombinant HIVs encoding beta-galactosidase (HIV-LacZ) has allowed the rapid detection of HIV breakthrough within 48 hours of viral infection. Further refinement of these systems and their use to determine the frequency and causes of breakthrough infection is the goal of the proposed Experimental Plan. The Experimental Plan and Budget have been modified in response to the Initial Review Groups Summary Statement (see Introduction to Revised Application). This project will be an extension of an ongoing collaborative effort between the Principal Investigator at the University of Pittsburgh and Roger K. Strair, Co-Principal Investigator, at Yale University (subcontract).